Stimulation of T cells requires two types of signals - one antigen specific, the other antigen independent. The antigen-independent signal (the costimulatory signal) can be provided by the interaction of B7 molecules with specific receptors on the surface of the T cell. Costimulatory molecules may therefore be useful adjuncts to vaccination. We have cloned and sequenced the rhesus macaque homologues of human B7-1 and B7-2. These molecules are approximately 93-95% homologous to their human counterparts, with amino acid substitutions found throughout the molecule. CHO cells stably expressing B7-1 or B7-2 were recognized by several murine anti-human monoclonal antibodies. To confirm that the rhesus B7 molecules have the same costimulatory function as their human counterparts, CHO cells stably expressing B7-1 or B7-2 were fixed by UV-psoralen treatment and used to stimulate CD4+ T cells that had been suboptimally stimulated with anti-rhesus CD3 monoclonal antibodies. Stimulation of rhesus CD4+ T cells with anti-CD3 mAb alone induced a 5-7 fold increase in T cell proliferation compared with the medium control. Addition of fixed CHO-B7-1 or B7-2 induced a 10-27 fold increase in T cell proliferation compared to anti-CD3 alone. Control vector transfected CHO cells did not induce T cell proliferation over background. T cell proliferation could be blocked by preincubation of CHO B7-1 or B7-2 with anti-human B7-1 or B7-2 antibodies. Rhesus B7-1 and B7-2 molecules were also able to induce human CD4+ T cells to proliferate. The proliferation is specific and can be totally abrogated with anti-human B7-1 or B7-2 monoclonal antibody. After stimulation of rhesus CD4+ T cells, RT-PCR analysis of cytokine production showed that B7-1 stimulated T cells express interleukin 2, 4, 7, 10 and INF-gamma and B7-2 stimulated T cells express interleukin 2, 10 and INF-gamma. These molecular clones of B7-1 and B7-2 will be utilized to enhance in vitro stimulation of cellular immune responses, and to boost immune responses induced by vaccination.